We recently found that the in vitro generation of cytolytic T lymphocytes (CTL) from mouse spleen cells requires added soluble factors under some circumstances. These include the: (1) generation of CTL specific for alloantigens when the antigen is on tumor cells and the responding spleen cells are from nonimmunized mice; (2) generation of CTL specific for syngeneic tumor antigens when the responding spleen cells are from nonimmunized mice; and (3) generation of antiviral CTL from precursors from immunized mice if certain viral peptides are used. We have begun to characterize the needed factors biochemically and have found that the factor needed for the allogeneic response can come from phorbol myristic acetate and Concanavalin A-stimulated ROM4 cells and from nonstimulated WEHI-3, AFSTL1 and AFSTL2 cells. The WEHI-3 factor is interleukin-3 which also functions in assisting the generation of antiviral CTL using peptide antigens. Our major objective is to purify and characterize the new factors from ROM4, AFSTL1, and AFSTL2 cells both biochemically and functionally. (HF)